issn : 2454-9150 immobilization and characterization of
TRANSCRIPT
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
609 | IJREAMV07I0274215 DOI : 10.35291/2454-9150.2021.0295 © 2021, IJREAM All Rights Reserved.
Immobilization and Characterization Of Protease And
Peroxidase From Crude And Purified Extracts Of Carica
papaya Seeds And Brassica oleracea Wastes
1Jaincy Jayan, N., 2Shobana, A., 3Sujatha, A., 4Anitha Subash.
1Msc Biotechnology, 2,3Assistant Professor, 4Professor & Head Of The Department Of
Biochemistry, Department Of Biochemistry, Biotechnology And Bioinformatics, Avinashilingam
Institute For Home Science And Higher Education For Women, Coimbatore, Tamil Nadu, India,
[email protected], [email protected], [email protected],
ABSTRACT - The organic solid wastes of high biodegradability contributing to pollution can be used as a source of bio-
valuable products since they are rich in bio-active compounds. One of the important biomolecules of diverse use is the
enzymes and these enzymes could be made stable, resistant, and efficient by immobilization. In this study, we isolated
enzymes; protease and peroxidase from the wastes of papaya (seeds) and cabbage (leaves). The crude and purified
extracts were immobilized by different methods and tested for their reusability and storage activity. Their residual
activity was studied and the samples were characterized based on pH and temperature. The comparative study of pure
and immobilized samples and as well as the effective kind of immobilization could be helpful for the proper selection of
samples to be used for applications.
KEYWORDS - Alginate- Chitosan, Agar- Agar, Immobilization, Fruit and Vegetable wastes, Protease, Peroxidase and
Sodium-Alginate.
I. INTRODUCTION
Our world is facing a major environmental catastrophe due
to improper solid waste management (SWM) [11]. In India,
approximately 0.4 million tons of solid waste is generated
annually. The enormous waste production is triggered by
the surge in industrialization and urbanization as a result of
the exponential population growth [21]. SWM faces a
downside in fulfilling the criteria of environmentally
effective, socially acceptable, and affordable management
[15]. More than 1.3 billion tons of food wastes are generated
per year according to FAO (Food and agricultural
organization of the United Nations). There are two main
categories of food wastes: animal wastes (diary, fisheries,
and meat processing industries) and plant wastes (roots,
cereals, fruits, and vegetables). Food waste reduction and
utilization can reduce negative environmental and
socioeconomic impacts [6]. The Vegetable and Fruit wastes
(VFW) are categorized based on their stages of production,
distribution and transportation, processing, retailing/supply,
and consumer level [16]. VFW cannot be subjected to
incineration due to its high moisture content, and when
disposed of as landfills they can produce a large amount of
leachate due to their high organic content [22].
These residual agricultural wastes are rich in bio-active
compounds like phenolic compounds and secondary
metabolites; hence its re-utilization does not limit its
application to food\feed additives, nutraceuticals, and
cosmeceuticals but also helps in the production of other
value-added products as well as a beneficial measure for the
environment [7]. Researchers used microbial methods to
successfully develop products like enzymes, bio-ethanol,
flavouring agents, etc from VFW [16]. The high substrate
specificity, easier production, and green chemistry became
why the process of bio-catalysis is applied to every sector
[20].
Proteases are one of the most common degradative enzymes
found in plants and animals [8]. The seeds of Carica papaya
that contribute to VFW are a rich source of proteases. These
are cysteine proteases among which are the most abundant
Papain-like cysteine proteases (PLCPs) [4]. The papaya
proteases have four types of cysteine proteases; papain
(<10%), glycyl endopeptidases III and IV (23-28 %),
chymopapain A and B (26-30 %), and caricain (14-26 %)
forming a total protein content of 70-90%. The proteases
have broad thermo-stability and specificity. The papain
belongs to the hydrolases group. They have an isoelectric
point at pH 8.75, a molecular weight of 23,406, and
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
610 | IJREAMV07I0274215 DOI : 10.35291/2454-9150.2021.0295 © 2021, IJREAM All Rights Reserved.
optimum catalytic activity at the range of pH 5-8 [5]. The
papain has applications in the medical field; against cancer,
inflammation, diabetes, etc. The papain from seeds is found
to have anthelmintic properties [4]. It is also used in the
clarification of beverages, as meat tenderizing agents, and
as a digestive aid [13].
Peroxidases are hemoproteins that are widely found in
nature. They play an important role in catalyzing the
reduction of H202 which is an end product of oxidative
metabolism [2]. The cabbage heads and leaves which form
the part of the VFW are rich in peroxidases. The cabbage
peroxidases are known to be a heterogenous heme protein
that possesses high heat stability, optimum activity in the
range of 6-8 pH, and are a good choice for effective
immobilization [2]. The Cabbage peroxidases have quite
versatile applications in different fields. They are efficient
for the construction of stable bio-electrocatalytic systems
for oxygen reduction, blanching procedures in the food
industry due to their heat stability [5]. and in the removal of
pollutants (phenol and dye) [14].
The extensive commercialization of enzymes has its limit
due to the expense, low reusability, and other dependent
parameters. These challenging factors can be tackled by
efficient immobilization; the confinement of enzymes to a
suitable solid matrix/support. The ideal matrix is chosen
based on the characteristics like inertness, physical strength,
stability, reusability, ability to enhance enzyme activity,
reduce product inhibition, prevent contamination, and as
well as be economical. Entrapment, adsorption, covalent
binding, and membrane confinement are the different
methods of immobilization [20].
II. MATERIALS and METHODS
1. Preparation of crude and purified homogenate
The papaya and cabbage wastes were collected from the
local market and cleaned.154 g of papaya seeds was
homogenized with 0.1 M phosphate buffer, pH 7.2, and
centrifuged at 4 ⁰C for 20 minutes at 5000 rpm and filtered
through Whatman No 1 filter paper. 200 g of cabbage
wastes were homogenized with 0.1M Tris HCl, pH 7.5. The
solution obtained was centrifuged at 4 ⁰C 20 minutes at 5000
rpm and filtered through Whatman No 1 filter paper to
obtain a clear solution. This filtrate was subjected to thermal
treatment at 65 ⁰C for 5 minutes, followed by cooling on ice
for 25 minutes to selectively remove contaminate traces of
other enzymes. These crude extracts were subjected to
Ammonium Sulphate precipitation (0-100% concentration),
followed by dialysis of the specific aliquots having a high
concentration of enzyme. Both the samples containing
enzymes were then used for immobilization.
2. Immobilization
2.1 Sodium-Alginate
This is one of the common methods of immobilization by
entrapment by the formation of Calcium-Alginate beads.
The sodium alginate mixture (2%) is mixed with enzyme
solution in the ratio of 1:1. Using a 10 ml syringe, this
mixture was added dropwise into 0.2M CaCl2. Left the
beads formed to harden for 20 minutes and washed with
deionized water for a couple of times followed by respective
buffers and stored at 10 ⁰C.
2.2 Alginate-Chitosan
200 mg of alginate was dissolved in 10 ml of the respective
buffers at 40 ⁰C for 30 minutes and brought to room
temperature to which liquid enzyme (0.5 mg/ml) was added
and stirred till a complete homogenate was obtained.
Another solution was prepared by mixing 500 mg of
chitosan in acetic acid (v/v, 2%) and heated at 50 ⁰C. 0.7 M
CaCl2 was added into this clear solution and stirred. In order
to form the beads, the Enzyme-Alginate mixture was added
to Chitosan-CaCl2 using a syringe and these beads were
washed using the buffer and stored at 10 ⁰C.
2.3 Agar-Agar
2 % agar was prepared in respective buffers by heating at
50 ⁰C. When the solution reaches room temperature equal
volume of enzyme solution was added and poured into
plates to solidify. The gels formed were cut into cubed
pieces, washed with buffer, and stored at 10 ⁰C.
3. Protease assay
The reaction was carried out in triplicates.1 ml of the
enzyme and 0.5 ml of casein was incubated for 10 minutes
and to which 5 ml of TCA followed by 30 minutes of
centrifugation at 5000 rpm was done. 2 ml of the
supernatant was mixed with 5 ml of Sodium Carbonate and
1 ml of Folin-ciocalteau and incubated for 10 minutes. The
colour developed was measured colorimetrically at 670 nm.
Enzyme activity was calculated from the formula,
Units/ml enzyme =
µmol tyrosine equivalents released x total vol. of the assay
(ml)
vol. of enzyme used (ml) x time of assay in minutes x
vol. used in colorimetric determination (ml)
4. Peroxidase Assay
The 100 µl of the enzyme was mixed with 3 ml buffer
followed by 50 µl guaiacol and 30 µl H2O2 and the colour
developed was read at 450 nm from time to time for 10
minutes.
Enzyme activity (Units/L) = (∆Abs ×Total assay
vol)/∆t×€×l× enzyme sample volume
5. Determination of reusability and storage
For the reusability test, the assays were repeated for
alternate days for up to 9 and 7 days for crude and purified
samples. For the storage test, the stored immobilized
samples were assayed on a period of 5 or 7 days. The
residual activity was recorded after each assay.
6. Characterisation based on pH and Temperature
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
611 | IJREAMV07I0274215 DOI : 10.35291/2454-9150.2021.0295 © 2021, IJREAM All Rights Reserved.
For pH, the reaction was carried out in triplicates at 5-9 pH
and for temperature, 10,30,50,70 C were taken. At those
particular conditions, the enzyme activity was recorded and
studied.
III. RESULTS
1. Immobilized Crude Protease
50 ml of crude was used to produce 43.12 g Sodium-
Alginate beads and 64 g of Agar-Agar gels. 23 g of
Alginate-Chitosan beads were made from 10 ml of the
extract. The beads are shown in figure 1.
Figure 1: a) Sodium-Alginate b) Alginate-Chitosan and c) Agar-Agar
1.1 Determination of reusability
The reusability of all the beads was checked during the
period of alternate 9 days. Figure 2 shows the gradual
decrease in the immobilized crude protease activity. The
enzymes entrapped in Sodium-Alginate beads showed a 100
% activity for the first day and 94, 75, 50, and 25 %
respectively for the 3rd, 5th, 7th and 9th day. This shows that
from the 3rd day, it showed a decline in activity by 25% till
the 9th day. For Alginate-Chitosan beads, the enzyme
activity showed a stable 100% activity till the 3rd day, then
it showed a gradual decline by 12-50 %, whereas in the case
of Agar-Agar gels the enzyme activity recorded were 100,
93, 73, 47 and 27 % till the 9th day.
Figure 2: Determination of reusability of immobilized crude protease
1.2 Determination of storage capacity
The enzyme activity of the stored beads and gels is shown
in figure 3. The activity was checked for the 1st ,7th, and 11th
day. The stored beads of Alginate-Chitosan showed a
constant 100% activity for 7 days and the curve declined by
12% by the 11th day. The enzyme activity was a 100, 94 and
56 % from the Sodium-Alginate beads. The Agar- Agar gels
had 100, 94 and 47 % enzyme activity. There-fore the
Sodium-Alginate beads and Agar-Agar gels showed a 6 %
decline by the 7th day and 44 % and 53 % respectively by
the 11th day.
Figure 3: Determination of storage capacity of immobilized crude
protease
1.3 Effect of pH
The pH stability was monitored using phosphate buffers of
varying pH 5, 6, 7, 8, and 9. Figure 4 represent the findings
of the pH stability. The Sodium- Alginate samples had
enzyme activity of 66, 83, 100, 83 and 83 % at pH 5, 6, 7, 8
and 9. Whereas it was 75, 83, 100, 92 and 92 % for the
Alginate- Chitosan samples and a 67, 89, 100, 89 and 89 %
for the Agar- Agar samples for the respective pHs. The
enzyme activity showed a peak in pH 7 compared to the
other ranges in all the three types of immobilized samples.
There was a slight decrease in pH 5 and 6 and the activity
remained constant through pH 8-9 for the Sodium-Alginate,
Alginate-Chitosan and Agar-Agar samples.
Figure 4: Effect of pH on immobilized crude protease
1.4 Effect of temperature
The temperature variation is depicted in figure 5. The
enzyme activity from all the beads and gels was tested at
temperatures 10 ⁰C, 30 ⁰C, 50 ⁰C, and 70 ⁰C. The enzyme
activity was dependent on the temperature. There was a
gradual increase in activity with the temperature in case of
all samples. The enzyme activity was 50, 67, 100, and 100
% at the respective temperatures for the Sodium-Alginate
samples and it was a 76, 82, 94 and 100 % for the Alginate-
Chitosan samples. The Agar- Agar samples showed an
enzyme activity of 50, 60, 90 and 100 % at temperatures 10
⁰C, 30 ⁰C, 50 ⁰C and 70 ⁰C. The lowest activity was shown
at 10 ⁰C and a stable increase was recorded through 30 ⁰C –
50 ⁰C. The optimum activity was shown at 70 ⁰C for all
kinds of immobilization.
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
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Figure 5: Effect of temperature on immobilized crude protease
2. Immobilization of Purified Protease
50 ml of the purified sample yielded 44 g and 60 g of
Sodium-Alginate beads and Agar-Agar gels and 10 ml
yielded 32g of Alginate-Chitosan beads as shown in figure
6.
Figure 6: a) Sodium-Alginate b) Alginate-Chitosan and c) Agar-Agar
2.1 Determination of reusability
The reusability data studied for all three types of
immobilization is given in figure 7. The enzyme activity
was checked for a period of alternate 7 days. For the first 3
days, the Alginate-chitosan beads released enzyme in a
constant amount and it showed 100% activity. The rate
decreased by 9% and 12% by the next 4 days. The enzyme
activity recorded for Sodium-Alginate samples were 100,
95, 77, and 64 % for the 1st, 3rd, 5th, and 7th day. The Agar-
Agar samples had 100, 95, 76, and 57 % of enzyme activity
for the respective alternate days. Hence by the 7th day the
enzyme activity decreased by around 30 and 40 % for the
Sodium-Alginate and Agar-Agar samples.
Figure 7: Determination of reusability of immobilized purified
protease
2.2 Determination of storage capacity
The storage capacity of the immobilized beads and gels is
depicted in figure 8. The enzyme activity remained linear
showing 100% of its effect for the stored Alginate-Chitosan
beads when checked for the 5th and 7th day. In the case of
Sodium-Alginate and Agar-Agar, the activity was 100%
when checked on the 5th day and decreased by 5% and 14%
respectively when checked on the 7th day since it showed a
residual activity of 95 and 90 %.
Figure 8: Determination of storage capacity of immobilized purified
protease
2.3 Effect of pH
The pH of the varying range tested is depicted in figure 9.
Phosphate buffers of pH 5,6,7,8, and 9 were used to record
the stability of the immobilized enzyme. There was a
gradual increase in the activity from pH 5-7, with the
highest activity at 7. After that, the activity started to decline
from pH 8-9. The enzyme activity was 90, 95, 100, 90 and
90 % for the Sodium-Alginate samples and 89, 89, 100, 94
and 94 % for Agar-Agar samples. The Alginate-Chitosan
samples had an enzyme activity of 87, 93, 100, 93 and 93 %
at pH 5, 6,7,8, and 9.
Figure 9: Effect of pH on immobilized purified protease
2.4 Effect of temperature
The thermal activity was recorded at temperatures 10 ⁰C, 30
⁰C, 50 ⁰C, and 70 ⁰C as shown in figure 10. There was a
stable linear increase in the activity for Alginate- Chitosan,
Sodium-Alginate and Agar-Agar samples. The optimum
activity was observed at temperature 70 ⁰C for all the
samples. The samples of Sodium-Alginate showed an
activity of 71, 86, 96 and 100 % and those of Agar-Agar had
83, 89, 92 and 100 % activity at those respective
temperatures. The enzyme activity was 81, 90, 96 and 100
% for the Alginate- Chitosan samples at 10 ⁰C, 30 ⁰C, 50 ⁰C
and 70 ⁰C.
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
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Figure 10: Effect of temperature on immobilized purified protease
3. Immobilization of Crude Peroxidase
The 50 ml of sample yielded 51 g and 62 g of Sodium-
Alginate beads and Agar-Agar gels respectively. 10 ml of
the sample yielded 22 g of Alginate-Chitosan beads (Figure
12).
Figure 12: a) Sodium-Alginate b) Alginate-Chitosan and c) Agar-Agar
3.1 Determination of reusability
The reusability of immobilized crude peroxidase was tested
alternatively for 9 days as shown in figure 13. The Alginate-
Chitosan and Sodium-Alginate samples showed 100%
activity for the first 3 days and the former showed a decline
by 5% whereas the latter by 18% by the 5th day. The
enzyme activity was 82, 75, and 56 %; 95, 80 and 70 %
during the 5th, 7th, and 9th day for Sodium-Alginate and
Alginate-Chitosan samples. The enzyme activity observed
for Agar-Agar was 100, 95, 79, 68, and 53 % for the
alternate 9 days. The Agar-Agar samples showed a decline
by 50 % by the 9th day.
Figure 13: Determination of reusability of immobilized crude
peroxidase
3.2 Determination of storage capacity
Figure 14 shows the storage capacity of the immobilized
crude samples for 11 days. The Alginate-Chitosan sample
showed a 100% activity for 7 days and declined by 5% on
the 11th day since it recorded a 95 % of enzyme activity.
The Sodium-Alginate and Agar-Agar samples showed
100% activity when checked on the 7th day and decreased
by 12% and 15% respectively for the 11th day. On the 11th
day the Sodium-Alginate and Alginate-Chitosan showed an
enzyme activity of 87 and 84 %.
Figure 14: Determination of storage capacity of immobilized crude
peroxidase
3.3 Effect of pH
The pH ranges of 5-9 were checked as shown in figure 15.
The tris HCl buffer of varying pH was used to check for
stability. The samples showed increased stability from 5-7
pH with its peak at pH 7. The activity declined for the pH
range 8-9. The Sodium-Alginate showed enzyme activity of
100 % at pH 7 and 85 % for both pH 8 and 9. At an acidic
pH of 5 it showed 92 %. The Alginate-Chitosan samples
showed an increasing activity of 89, 95 and 100 % from pH
5-7 and a 89 % of activity at 8 and 9 pH. The Agar-Agar
gels showed an enzyme activity of 80, 93, 100, 93 and 80 %
at the respective pHs.
Figure 15: Effect of pH on immobilized crude peroxidase
3.4 Effect of temperature
The thermal stability was tested at temperatures at 10 ⁰C, 30
⁰C, 50 ⁰C, and 70 ⁰C is shown in figure 16. The activity
increased gradually from 10 ⁰C-50 ⁰C with its peak at 50 ⁰C.
At 70 ⁰C, the activity started decreasing by 20-40 % in the
case of all the samples. The Sodium-Alginate samples
showed 86, 93, and 100 % activity and the Alginate-
Chitosan showed 84, 95, and 100 % activity at temperatures
10 ⁰C, 30 ⁰C, and 50 ⁰C. The Agar- Agar samples had an
activity of 87, 94, and 100 % till 50 ⁰C. At 70 ⁰C, the activity
recorded was 71, 65, and 81 % for the Sodium-Alginate,
Alginate-Chitosan and Agar- Agar samples.
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
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Figure 16: Effect of temperature on immobilized crude peroxidase
4. Immobilization of Purified Peroxidase
50 ml of the purified sample yielded 50 g and 58 g of
Sodium-Alginate beads and Agar-Agar gels and 10 ml
yielded 30 g of Alginate-Chitosan beads as shown in figure
17
Figure 17: a) Sodium-Alginate b) Alginate-Chitosan and c) Agar-Agar
4.1 Determination of reusability
The reusability of the immobilized pure peroxidase was
tested for 7 days as shown in figure 18. From the graph, it
is evident that all the samples showed maximum activity till
the 3rd day. For the next 4 days the activity showed a
gradual decline of 8-11%. The enzyme activity for Sodium-
Alginate samples was 92 and 89 % and for Agar-Agar
samples it was 93 and 90 % for the 5th and 7th day. The
Alginate-Chitosan samples displayed an enzyme activity of
93 and 90 % on the last two alternate days.
Figure 18: Determination of reusability of immobilized purified
peroxidase
4.2 Determination of storage capacity
The storage capacity of the stored samples of immobilized
pure peroxidase is depicted in figure 19. It shows a stable
and uniform enzyme activity of 100 % for 7 days in the case
of the Alginate-Chitosan samples. For the Agar-Agar
samples, it showed a 100 % activity for the 5th day and
declined by 4% on the 7th day since it recorded an enzyme
activity of 96 %. The Sodium-Alginate samples also
showed a 100 and 96 % activity on the 5th and 7th day.
Figure 19: Determination of storage capacity of immobilized purified
peroxidase
4.3 Effect of pH
The pH range of 5-9 was tested as shown in figure 20. The
samples show a gradual increase from pH 5-7 with peak
activity at pH 7 and a decline from 7-9 pH. The Sodium-
Alginate showed an enzyme activity of 92, 96, 100, 92 and
88 % at the respective pHs (5, 6, 7, 8 and 9). The Agar-Agar
samples showed a 96 % activity at pH 5 and 6; 92 % at pH
8 and 9; and a 100 % at pH 7. The enzyme activity recorded
for the Alginate-Chitosan samples were 93, 96, 100, 93, and
93 %. This shows the peak activity at pH 7.
Figure 20: Effect of pH on immobilized purified peroxidase
4.4 Effect of temperature
The thermal stability was recorded at temperatures 10 ⁰C,
30 ⁰C, 50 ⁰C, and 70 ⁰C as shown in figure 21. The activity
showed a constant increase from 10-50 ⁰C and steeps down
at 70 ⁰C. The enzyme activity was 88 and 96 %; 86 and 96
%; 89 and 96% at temperatures 10 ⁰C and 30 ⁰C for the
Sodium-Alginate, Alginate-Chitosan and Agar-Agar
samples. All the beads and gels recorded a 100 % activity at
50 ⁰C. At 70 ⁰C, the activity recorded was 81, 79, and 74 %
for the Sodium-Alginate, Alginate-Chitosan and Agar-Agar
samples.
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Figure 21: Effect of temperature on immobilized perified peroxidase
IV. DISCUSSION
The immobilized crude protease of the Alginate-Chitosan
method retained 47% of residual activity by the 9th day. The
Sodium-Alginate and Agar-Agar immobilization left a
residual activity of 25 and 27% of crude protease. This
shows the reusability of the enzyme was effective for
Alginate-Chitosan immobilization compared to the other
two methods. The decrease in enzyme activity might be due
to the frequent thawing and change of buffers. [19] stated
that the residual activity was 50 % up to 10th use when
protease immobilized on Glutaraldehyde-Activated
Chitosan matrix was used. For the storage capacity, the
enzyme activity was 88 % on the 11th day for Alginate-
Chitosan immobilized protease and 56 and 47 %
respectively for Sodium-Alginate and Agar-Agar methods.
This proves the storage time of much effective with minimal
loss of activity is possible with Alginate-Chitosan
immobilization of crude protease. The activity remained
constant through pH 8-9. The optimum pH was 7 for
immobilized plant protease [17]. The stability of pH is
achieved by all the types of immobilization since the
difference between the optimum pH and the consecutive
ones is not more than 30 %. The pH stability is high for
immobilized proteases compared to soluble proteases [19].
The optimum temperature range of immobilized proteases
is around 70 ⁰C [9]. The broad range of thermal stability
shows that immobilization could be an effective and ideal
method to obtain high yield in industrial applications.
The residual activity of immobilized purified protease was
much higher compared to the immobilized crude protease.
They showed a 78, 64 and 57 % activity by the 7th day for
Alginate-Chitosan, Sodium-Alginate, and Agar-Agar
respectively. In another study, the activity of immobilized
protease and purified protease was 70 % for 10th reuse and
65 % for the 6th cycle [10] and [12]. This shows that
purified protease could be effectively reused when
immobilized on the Alginate-Chitosan matrix followed by
the other two methods. In my study, the residual activity
was 100,95 and 90% on the 7th day when the pure protease
was immobilized on Alginate-Chitosan, Sodium-Alginate,
and Agar-Agar for the storage capacity assay. This activity
is twice greater than that was recorded for the crude protease
making the sample ideal for storage for further use. The
chances of decline in the activity after the tested time period
could be only by 4 % or less if stored under proper
conditions. According to [12], the storage activity decreased
by 12 % after 10 days. The immobilization protected the
enzyme enabling a broad range of pH stability. The
temperature of 70 ⁰C being optimum is supported by [9].
The immobilized pure protease shows a broad range of
thermal stability.
The immobilized crude peroxidase showed residual activity
of 70,56, and 53% for the 9th day for Alginate-Chitosan,
Sodium-Alginate, and Agar-Agar methods. The chitosan
method of immobilization has high reusability compared to
others. Immobilization of peroxidase from a different
source showed 75 % reusability in another study [1]. The
stored samples showed a residual activity of 95,87 and 84%
for Alginate-Chitosan, Sodium-Alginate, and Agar-Agar
respectively when checked for the 11th day. It is evident that
the Chitosan matrixes retained the enzyme activity
effectively compared to the other two. [1] stated the storage
stability of immobilized peroxidase dropped to 63 % after 8
weeks. The optimum activity between pH 6-7 is supported
by [3]. This shows the immobilized crude peroxidase
showed activity at a broad with only a 5-10 % difference
compared to the optimum pH. The broad range of thermal
stability is an advantage for industrial applications.
The residual activity of immobilized purified peroxidase
was higher than immobilized crude peroxidase as it showed.
The enzyme activity was 90,90 and 89% for Alginate-
Chitosan, Agar-Agar, and Sodium-Alginate methods. This
shows both Chitosan and agar matrixes were efficient in
maintaining the enzyme activity [18]. The storage capacity
of immobilized purified peroxidase is higher than
immobilized crude samples. They showed a residual
activity of 100,96 and 96 % on the 7th day for Alginate-
Chitosan, Sodium-Alginate, and Agar-Agar samples. The
Chitosan matrixes retained the enzyme activity efficiently,
followed by the other two types. [18] stated that the storage
capacity of immobilized peroxidases relatively higher than
peroxidases. The optimum pH activity was observed in the
range of 6-7 [1]. The activity was quite higher than the
immobilized crude peroxidase. Even though it shows a
decline from the optimum activity, there is a broad range of
stability. The high activity of cabbage peroxidase at 50 ⁰C
is supported by [3]. The immobilized enzyme shows
significant stability over a range of temperatures. At very
high temperatures the enzyme behaves inversely
proportional to the change.
The methods of immobilization of protease and peroxidase
were compared as shown in figures 22 & 23. Among the
three types (Sodium-Alginate, Alginate-Chitosan, and
Agar-Agar), the Alginate-Chitosan immobilized crude and
purified samples showed promising results when compared
to the Sodium-Alginate and Agar-Agar immobilization. The
enzyme activity was high (as evident in the given figures)
when the reusability and storage capacity were checked
using the same making it the ideal form of immobilization
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
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to opt. When compared to the Alginate-Chitosan (100 %)
the activity for Sodium-Alginate and Agar-Agar for
immobilized crude and purified protease is 94 and 88 %; 68
and 65 % whereas for the immobilized crude and purified
peroxidase is 80 and 95 %; 90 and 97 % respectively. The
Sodium-Alginate showed the second highest activity for
immobilized protease samples whereas it is the Agar-Agar
method for the immobilized peroxidase samples. It could be
the stronger bonding resulting in greater stability compared
to the latter. The Agar-Agar showed relatively less activity
and stability for protease. It was expected of it because the
gels started to break down or dissolve after a few
experiments. Chitosan mixed matrixes were much stable
and efficient since they showed promising results when
tested under varying pH and temperatures. Future
experimentations can be carried out using different
concentrations of the all these matrices to increase the
efficiency furthermore.
Figure 22: Comparison of Immobilized crude and purified Protease
based on its activity
Figure 23: Comparison of Immobilized Crude and Purified Peroxidase
based on its activity
V. CONCLUSION
Immobilization by different methods helps to understand
the interaction between enzymes and the matrices. The
immobilized purified samples exhibited high enzyme
activity, reusability, and storage with a thermal and pH
stability compared to the immobilized crude samples. The
reusability assay of the immobilized crude and purified
protease showed that the enzyme activity was 53 % by the
9th day and 78 % by the 7th day for the immobilized crude
and purified samples of Alginate-Chitosan methods which
was the highest compared to the Sodium-Alginate and
Agar-Agar methods. The stored samples of immobilized
crude and purified protease had 88 and 100 % activity on
the 11th and 7th day. The stored Sodium-Alginate and Agar-
Agar samples of immobilized purified protease also showed
high level of activity of 95 and 90 % on the 7th day. Both the
immobilized crude and purified protease had optimum pH
of 7 and a temperature of 70 ⁰C. This activity showed only
little variation at other ranges of the chosen parameters
proving the stability range of the immobilized samples. In
case of peroxidase, the reusability assay showed 70 % and
90 % activity for the Alginate-Chitosan samples of
immobilized crude and purified enzyme on the 9th and 7th
day. The immobilized purified samples of peroxidase
showed 89 and 90 % activity for the Sodium-Alginate and
Agar-Agar samples on the 7th day. The stored immobilized
crude and purified peroxidase of Alginate-Chitosan method
had 95 and 100 % activity when checked on the 11th and 7th
day. All the peroxidase samples had an optimum pH of 7
and temperature of 50 ⁰C. These findings conclude the
effective method of immobilization as well as the source
needed to be used. The immobilized forms of these enzymes
can reduce the cost and increase the efficiency of product
formation with minimal waste since both protease and
peroxidase finds application in wide variety of fields. The
immobilized purified protease can be applied for detergent
industry, to remove different types of stain, since the free
enzymes are susceptible to denaturation in the presence of
other chemicals like oxidizing agents. These immobilized
proteases are well protected within a porous matrix hence
they will not be destroyed by other cells of the body hence
can also be used to study and treat cancer and diabetes. The
immobilized peroxidases also showed a promising activity
at different ranges of pH and temperature hence these,
especially the purified ones will be able to remove effluents
and dyes from the polluted waters since it can withstand the
harsh conditions of wastewater treatment.
ACKNOWLEDGEMENT
I owe a special tribute to God Almighty for the opportunity
given to take up to complete my work successfully. I
express my deep sense of gratitude to all my higher
authorities, Dr. (Thiru) S.P. Thiyagarajan, chancellor,
Former Chancellor Dr. (Thiru) P.R. Krishna Kumar, (Late), Dr. Premavathy Vijayan, Vice Chancellor, Dr. S. Kowsalya,
Registrar, Dr. A. Vijayalakshmi, Dean, School of
Biosciences, Professor and Head, Department of Botany, Dr. P.R. Padma (Late), former Dean and Head, Department
of Biochemistry, Biotechnology and Bioinformatics, and
Dr. G.P. Jeyanthi, Director, Research and Consultancy. I
express my gratitude to my co-authors Dr. A. Shobana,
Assistant Professor, Department of Biochemistry,
Biotechnology and Bioinformatics, Dr. Sujatha, Assistant
Professor, Department of Biochemistry, Biotechnology and
Bioinformatics, and Dr. Anitha Subash, Professor and
Head, Department of Biochemistry, Biotechnology and
Bioinformatics, of Avinashilingam Institute for Home
Science and Higher Education for Women for their
immense support. I submit my sincere thanks to all The
Staff Members of the Department. I express my sincere
International Journal for Research in Engineering Application & Management (IJREAM)
ISSN : 2454-9150 Vol-07, Issue-02, MAY 2021
617 | IJREAMV07I0274215 DOI : 10.35291/2454-9150.2021.0295 © 2021, IJREAM All Rights Reserved.
gratitude to my parents Nancy.J and Jayan.J for their moral
support & guidance throughout my study. I express my
sincere heart bound thanks to my friends and seniors,
Department of Biochemistry, Biotechnology and
Bioinformatics, for giving an affectionate advice,
unconditional love and incredible supports for the
completion of my project work.I acknowledge the
contribution of all other unseen hands during the course of
the study for help rendered in the successful completion of
the study.
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